Gene delivery for therapy or other purposes is well-known, particularly for the treatment of diseases such as cystic fibrosis and certain cancers. The term refers to the delivery into a cell of a gene or part of a gene to correct some deficiency. In the present specification the term is used also to refer to any introduction of nucleic acid material into target cells, and includes gene vaccination and the in vitro production of commercially-useful proteins in so-called cell factories.
Cell delivery systems fall into three broad classes, namely those that involve direct injection of naked DNA, those that make use of viruses or genetically modified viruses and those that make use of non-viral delivery agents. Each has its advantages and disadvantages. Although viruses as delivery agents have the advantages of high efficiency and high cell selectivity, they have the disadvantages of toxicity, production of inflammatory responses and difficulty in dealing with large DNA fragments.
Non-viral gene delivery systems are based on the compaction of genetic material into nanometric particles by electrostatic interaction between the negatively charged phosphate backbone of DNA and cationic lipids, peptides or other polymers (Erbacher, P. et al, Gene Therapy, 1999, 6, 138-145). Various mechanisms for the action of these species have been suggested. An early suggestion was that membrane fusion between liposome and cell membrane occurs. More recently, endocytosis of intact complexes has been proposed. Complexes formed between the nucleic acid and the lipid become attached to the cell surface, then enter the cell by endocytosis. They then remain localised within a vesicle or endosome for some time and the nucleic acid component is released into the cytoplasm. Migration of the nucleic acid into the nucleus may then occur some time later, where a gene encoded by the nucleic acid may be expressed. Gene expression in the nucleus involves transcribing DNA into RNA and then translating that into protein.
The use of lipids, rather than viruses, for this purpose can result in lower toxicity, reduced cost, reasonably efficient targeting, and the ability to deal with large fragments of nucleic acid material. Unfortunately, lower transfection efficiencies have been noted.
Known complexes for gene delivery include “LID complexes”. As used herein, the term “LID complex” represents a complex comprising a lipid, an integrin- (or other receptor-) binding peptide and DNA. LID complexes achieve transfection via an integrin-mediated pathway; they do not necessarily need to have an overall positive charge so undesirable serum interaction can be reduced. The lipid component shields both DNA and, to a degree, the peptide component from degradation, endosomal or otherwise. The peptide component can be designed to be cell-type specific or cell-surface receptor specific. For example the degree of integrin-specificity can confer a degree of cell specificity to the LID complex. Specificity results from the targeting to the cell-surface receptors (for example integrin receptors), and transfection efficiencies comparable to some adenoviral vectors can be achieved (Hart et al., “Lipid-Mediated Enhancement of Transfection by a Nonviral Integrin-Targeting Vector”, Hum. Gene Ther., 9: 575-585 (1998); Harbottle et al., “An RGD-Oligiolysine Peptide: A Prototype Construct for Integrin-Mediated Gene Delivery”, Hum. Gene Ther., 9(7): 1037-1047 (1998); and Jenkins et al., “An Integrin-targeted Non-viral Vector for Pulmonary Gene Therapy” Gene Therapy, 7, 393-400 (2000). Peptides that target human airway epithelial cells have been reported (WO02/072616). Peptides that dendritic cells have been reported (WO2004/108938).
The components of a LID complex associate electrostatically to form a Lipid/Peptide vector complex a so-called lipopolyplex type of vector (Hart et al. (1998) op. cit.; Meng et al., Efficient transfection of non-proliferating human airway epithelial cells with a synthetic vector system, J. Gene Med., 6, 210-221 (2004); Parkes et al., High efficiency transfection of porcine vascular cells in vitro with a synthetic vector system, J. Gene Med., 4, 292-299 (2002)). Lipid/peptide vectors transfect a range of cell lines and primary cell cultures with high efficiency and low toxicity: epithelial cells (40% efficiency), vascular smooth muscle cells (50% efficiency), endothelial cells (30% efficiency) and haematopoietic cells (10% efficiency). Furthermore, in vivo transfection of bronchial epithelium of mouse has been demonstrated (Jenkins et al., Formation of LID vector complexes in water alters physicochemical properties and enhances pulmonary gene expression in vivo, Gene Ther., 10, 1026-1034 (2003), rat lung (Jenkins et al. (2000) op. cit.) and pig lung (Cunningham et al., Evaluation of a porcine model for pulmonary gene transfer using a novel synthetic vector, J. Gene Med., 4, 438-446 2002)) and with efficiency comparable to that of an adenoviral vector (Jenkins et al. (2000) op. cit.).
A peptide for use in such LID complexes or lipid/peptide complexes must have two functionalities: a “head group” containing a cell surface receptor- (for example integrin-) recognition sequence and a “tail” that can bind DNA non-covalently. Known peptides in which these two components are covalently linked via a spacer in a way that does not interfere with their individual functions include peptides in which the “tail” is a polycationic nucleic acid-binding component, such as peptide 6 as described in WO96/15811.
Initial experiments involving LID complexes including such peptides have indicated insufficiently high transfection properties by the systemic, or intravenous, route of delivery. The likely problem, as described for other polycationic vectors is association of the vector with serum proteins and red cell membranes leading to poor solubility and rapid clearance of the vector by the reticuloendothelial systems (Dash, P. R., Read, M. L., Barrett, L. B., Wolfert, M. A., Seymour, L. W. (1999) Gene Therapy 6, 643-50). Vectors that have displayed some transfection activity by systemic administration have been effective largely in first-pass capillary beds of organs such as the liver and lung (Fenske, D. B., MacLachlan, I., Cullis, P. R. (2001). Curr Opin Mol Ther 3, 153-8). While such non-specific transfection activity may have some therapeutic applications, safe clinical use for specific applications demands vectors with far greater target specificity.
Regarding the lipid component of the LID complexes, cationic lipids for such a use were developed by Felgner in the late 1980s, and reported in Proc. Natl. Acad. Sci. USA 84, 7413-7417, 1987. A patent to Felgner et al that may be referred to is U.S. Pat. No. 5,264,618. Felgner developed the now commercially-available cationic liposome known by the trademark “Lipofectin” which consists of the cytofectin, DOTMA and the neutral lipid DOPE in a 1:1 ratio. Various other cationic liposome formulations have since been devised, most of which combine a synthetic cationic cytofectin and a neutral lipid. Cytofectins are positively charged molecules having a cationic head group attached via some spacer to a hydrophobic tail. In addition to the DOTMA analogues, there may be mentioned complex alkylamine/alkylamides, cholesterol derivatives, and synthetic derivatives of dipalmitol, phosphatidyl-ethanolamine, glutamate, imidazole and phosphonate. A review of these materials, and of the mechanisms by which they operate, may be found in Angew. Chem. Int. Ed. 37, 1768-1785, 1998. However, cationic vector systems vary enormously in their transfection efficiencies in the presence of serum, which clearly impacts on their potential uses for in vivo gene therapy.
WO 2005/117985 describes lipids comprising one or more polyethylene glycol (PEG) groups (i.e. PEGylated lipids) and shows that such PEGylated lipids display benefits over lipids without PEG groups (i. e. non-PEGylated lipids). In particular, the problem of rapid clearance of lipids by the reticuloendothelial system caused by their binding to plasma proteins and vector aggregation may be ameliorated by shielding the vectors with polymeric PEG moieties. However, PEGylation often leads to greatly reduced transfection efficiency, and there remains a need for lipids which are not rapidly cleared by the reticuloendothelial system, but display satisfactory transfection efficiency.
Non-viral gene therapy vectors have been the subject of recent reviews: Hart, S. L., (2005) Current Drug Delivery, 2, 1-6; Kostareloa, K. and Miller, A. D., (2005) Chem. Soc. Rev., 34, 970-994.